The fraction of dead cells was quantified by flow cytometry (FACS Calibur, Becton Dickinson, Heidelberg, Germany; FL-2) of PI–stained cells as described previously [6 (link),8 (link),9 (link)]. Cells were incubated for 30 min in the dark with PI (10 μg/mL, Thermo Scientific, Waltham, MA, USA) in PBS and measured within 1 h.
To quantify cellular ROS production, cells were stained for 30 min at 37 °C with 5 μM Dihydroethidium (DHE) in RPMI 1640 medium (Molecular Probes/Invitrogen, Carlsbad, CA, USA). DHE-positive cells were detected by flow cytometry (BD FACS Calibur, Becton Dickinson; FL-2). ROS-production was evaluated by quantification of the fraction of DHE-positive cells (at least 10,000) with higher fluorescence.
To quantify cellular ROS production, cells were stained for 30 min at 37 °C with 5 μM Dihydroethidium (DHE) in RPMI 1640 medium (Molecular Probes/Invitrogen, Carlsbad, CA, USA). DHE-positive cells were detected by flow cytometry (BD FACS Calibur, Becton Dickinson; FL-2). ROS-production was evaluated by quantification of the fraction of DHE-positive cells (at least 10,000) with higher fluorescence.
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